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Differential susceptibility to the tracheal mite Acarapis
woodi between Apis cerana and Apis mellifera
Yoshiko Sakamoto, Taro Maeda, Mikio Yoshiyama, Jeffery S. Pettis
To cite this version:
Differential susceptibility to the tracheal mite
Acarapis
woodi between Apis cerana and Apis mellifera
Yoshiko S
AKAMOTO1,Taro M
AEDA2,Mikio Y
OSHIYAMA3,Jeffery S. P
ETTIS41National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan 2Institute of Agrobiological Sciences, NARO, 1-2 Ohwashi, Tsukuba, Ibaraki 305-0851, Japan 3Institute of Livestock and Grassland Science, NARO, 2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan
4USDA-ARS Bee Research Laboratory BLDG. 306 BARC-E, Beltsville, MD 20705, USA
Received 14 January 2016– Revised 25 May 2016 – Accepted 4 July 2016
Abstract– In Japanese honey bees Apis cerana japonica , infestations of the tracheal mite Acarapis woodi have spread rapidly over the mainland of Japan, causing damage and the collapse of colonies. Meanwhile, infestations by mites in Apis mellifera have hardly been observed in Japan. In this study, we assessed and compared the susceptibility of the two species, A. cerana and A. mellifera , using an inoculation assay. We found that migrating female mites entered the tracheae of more newly emerged bees in both species but more frequently in A. cerana than in A. mellifera . Hence, the higher susceptibility in A. cerana is proposed as a factor causing the explosive epidemic of tracheal mites in only A. cerana in Japan. Moreover, we compared grooming behaviors between the two bee species using an observation assay as a preliminary experiment, although the bees were not exposed to the presence of tracheal mites. From these observations, the frequency of autogrooming (self-grooming) on the thorax in A. cerana was lower than that in A. mellifera . The difference in susceptibility to the mite between these two species may be due to the difference in grooming behavior frequency.
Asian honey bee / European honey bee /Acarapis woodi / susceptibility / grooming behavior
1. INTRODUCTION
The tracheal mite Acarapis woodi , which was
first associated with a condition that caused
con-siderable colony mortality in Apis mellifera on the
Isle of Wight, England, in the early 1900s (Rennie
1921
), has spread all over the world with the
ex-ception of Sweden, Norway, Denmark, and
Aus-tralia (Sammataro et al.
2000
). Both larval and
adult mites feed on bee hemolymph in the tracheal
tubes of adult bees (Pettis and Wilson
1996
), and
mated females leave the trachea and move to new
callow bees to begin a new infestation (Sammataro
and Needham
1996
). Mites are more successful in
moving to new bees at night when the adult bees
are not as active (Pettis et al.
1992
). Heavy mite
infestation results in colony losses by decreasing
longevity (Bailey and Lee
1959
), low honey
pro-duction (Eischen et al.
1989
), and the inability to
thermoregulate in cool climates (Eischen
1987
;
McMullan and Brown
2009
), due to damage of
the tracheal system. Major sources of the spread of
tracheal mites between colonies could be the
drifting of infested bees to adjacent hives and
swarming (Eckert
1961
).
In 2010, infestations by the tracheal mite
A. woodi were first recorded in Apis cerana
japon-ica in Japan (Ministry of Agriculture, Forestry and
Fisheries
2011
). In Japan, there are two Apis
spe-cies, the Japanese honey bee A. cerana japonica
which is a native subspecies existing only in Japan
and the western honey bee A. mellifera which is a
non-native species now found virtually worldwide
(Ruttner
1988
). Mite infestations have spread
Corresponding author: Y. Sakamoto, sakamoto.yoshiko@nies.go.jp Manuscript editor: Peter Rosenkranz
rapidly across a wide range of mainland Japan in
A. cerana japonica (Maeda
2015
), causing heavy
damage and the collapse of colonies during winter
(Maeda and Sakamoto
2016
). Meanwhile, mite
in-festations in A. mellifera have hardly been observed
in Japan (Kojima et al.
2011
; Maeda
2015
), despite
the fact that foragers of A. mellifera (Sasaki
1999
)
and A. cerana (our observation, unpublished) enter
each other
’s hives to steal honey, thus mixing
spe-cies. In recent years in A. mellifera , mite infections
are not epidemic or problematic, even in places once
heavily mite-infested (Wilson et al.
1997
).
So why does only A. cerana japonica suffer
from the mites in Japan? To understand this would
be useful information for the conservation of
A. cerana japonica. However, at this time the
fac-tors are not clear and could be multifactorial. One
potential factor is the use of miticides in hives of
A. mellifera . A natural ectoparasitic mite, Varroa
destructor , of A. cerana has shifted to A. mellifera
and is present on A. mellifera almost worldwide
(Anderson and Trueman
2000
; Solignac et al.
2005
). In Japan, two miticides, fluvalinate and
amitraz, are used only in hives of A. mellifera
which have no effective defense against the Varroa
mite. However, it is not clear how effective these
Varroa mite treatments are for reducing tracheal
mite infestations (Scott-Dupree and Otis
1992
),
even though there have been some positive reports
in lab experiments (Eischen et al.
1986
; Pettis et al.
1988
). Another possibility is genetic resistance to
the mite in A. mellifera . Some genetic strains of
A. mellifera resist damaging infestations of the
mites (Danka
2001
), and the resistance is linked to
allogrooming and autogrooming behaviors (Pettis
and Pankiw
1998
; Danka and Villa
2003
). Hence,
A. mellifera has likely evolved grooming behaviors
against the mite, which at least partially explains the
reduced mite damage in A. mellifera . Mite
infesta-tion mechanisms in A. cerana need to be studied,
and a comparison of mite susceptibility between
A. cerana and A. mellifera is needed.
In this article, we assessed and compared the
susceptibility of the two Apis species to tracheal
mites. The purpose of experiment 1 was to
deter-mine age susceptibility of each bee species over the
first 4 days of adult life using an inoculation assay.
Experiment 2 also used an inoculation assay to
compare the susceptibility of 0-day-old bees
(newly emerged bees, known to be most
suscepti-ble in A. mellifera ) between the two species. In
addition, we conducted an experiment to compare
the grooming behavior between the two species by
using an observation assay (experiment 3).
2. MATERIALS AND METHODS
2.1. Honey bee colonies
We utilized bee colonies of A. cerana japonica and A. mellifera , which were kept at the National Institutes for Environmental Study (NIES), Institute of Agrobiological Sciences, NARO (NIAS), Institute of Livestock and Grassland Science, NARO (NIRGS), and private apiaries in Tsukuba City, Ibaraki Prefecture, Ja-pan. We did not administer any Varroa treatments at least a half year before starting the experiments in all A. mellifera colonies. All laboratory assays were con-ducted at NIES, and a field assay was concon-ducted at NIAS. Experiments 1 and 2 were undertaken in November 2014. Experiment 3 was conducted in August 2015.
2.2. Preparation of host and target bees for
inoculation experiments 1 and 2
We conducted inoculation assays by putting intact live bees and mite-infested bees together under laboratory and field conditions. We refer to the mite-infested bees as Bhost^ bees and the intact bees to be tested as Btarget^ bees (Gary and Page1987). We collected foragers from a heavily infested bee hive of A. cerana japonica at the hive entrance and used them as host bees. We had confirmed a 100 % infestation rate in the foragers 1 month earlier. To obtain target bees, combs containing emerging bees from each colony were taken to the laboratory and kept in cages in an incubator to allow for adult bee emergence (35 °C, 55 % RH). Emerged bees (0–24 h old) were removed from the combs and marked with paint (Paint Marker, Mitsubishi, Japan) on their abdomen to distinguish age and colony source. All bees for these experiments, except host bees in a hive experiment, were narcotized by CO2
for 30 s before starting, to avoid rejection and to mix the bees thoroughly, and a preliminary experiment confirmed that there was no bias of proportional distribution between bee species. Host and target bees from the same colony were not used within an experiment.
2.3. Experiment 1
—susceptibility of bees
less than four days old
The purpose of experiment 1 was to assess the age-related susceptibility of each bee species from 0 to 3 days of age, not to compare the level of susceptibility between bee species. The mite ordinarily rarely enters the tracheae of the bees older than 4 days of age (Lee
1963; Gary et al.1989; Phelan et al.1991). Target bees of four different ages, <24, 24–48, 48–72, and 72–96 h (or 0, 1, 2, and 3 days of age) were prepared for each assay of bee species. Numbers of target bees of A. cerana and A. mellifera were 150 (34, 36, 40, and 40 with 0–3 days of age) and 150 (39, 40, 37, and 34 with 0–3 days of age), respectively. One- to 3-day-old target bees were maintained with sugar water (50 % v /v ) in a dark incubator at 32 °C and 55 % RH until used in the experiment. On November 10, the target bees of A. cerana and A. mellifera were separately put into cages (15 cm × 15 cm × 15 cm; BugDorm, MegaView Science, Taichung, Taiwan). The cages for A. cerana and A. mellifera are referred to as cages I and II and were maintained under dark conditions at 32 °C and 55 % RH. We put 47 and 52 individuals in cages I and II as host bees in conjunction with adding the target bees, respectively. The bees were provided ad libitum with sugar water (50 % v /v ) via a plastic petri dish (2 cm × 9 cm diameter) with three holes (8 mm diam-eter) on the bottom of the cage. A piece of comb (7 cm × 4 cm × 3 cm) was put on a wire and wood stand (6 cm × 12 cm × 4cm) after freezing at−20 °C for 24 h to kill wax moths. Figure1shows the experimental layout with the cage. We choose 7 days as the test period based on the evidence that the first adult mites to mature are the males after 11–12 days (Pettis and Wilson1996). Hence, after 7 days, the only adult mites present in the target bees are the founding females. The dead bees were removed from the cage, segregated, and counted. The live bees were put in a freezer and kept at−20 °C for 24 h, and then bees were segregated into host or target bee categories and counted. The target bees were also stored at−20 °C to await dissection.
2.4. Experiment 2
—interspecies comparison
of susceptibility
Inoculation assays were conducted twice in the same cages as in experiment 1 (15 cm × 15 cm × 15 cm; BugDorm, MegaView Science, Taichung, Taiwan) and
once in a Langstroth hive under field conditions. In this experiment, we used 0-day-old intact bees of both A. cerana and A. mellifera together as target bees to eliminate any unexpected differences among experi-mental setups. We started the cage experiment (cages I and II) on November 10 and 12, respectively. The total number of introduced host and target bees is shown in Table I. Four and three colonies of A. cerana and A. mellifera , respectively, were used to supply target bees. The experimental procedure of cage inoculation assay was the same as in experiment 1. For the hive inoculation assay, we introduce 0-day-old target bees of A. cerana and A. mellifera into a heavily infested hive in which outside workers were shown to be 100 % infested. The hive assay was performed beginning on November 13. The hive was estimated to have more than 3000 workers during that period. After 7 days, the target bees were removed from the hive using forceps. The storage method for bees was the same as in exper-iment 1 above.
2.5. Dissection technique
The bees were dissected using a modification to the classic technique of removing the head and thoracic collar as described by Lorenzen and Gary (1986). The prothoracic tracheae were carefully re-moved without crushing the end and placed on a double-sided tape placed on a glass slide. Using a stereomicroscope (M205C; Leica Microsystems GmbH, Wetzlar, Germany) at ×60–160 magnifica-tion, each trachea was opened using a microneedle. The number of adult mites of each sex, along with the number of larvae and eggs, was counted and recorded. This method mainly follows Mcmullan and Brown (2005).
2.6. Experiment 3
—interspecies comparison
in the frequency of grooming behaviors
same case to fill the comb. All bees for this experiment were narcotized by CO2for 30 s before starting to avoid
rejection between colonies in the observation hive. The observation hive was maintained at 25 °C in a labora-tory and covered with a cloth except during the obser-vations. The observations were made between 8:00 and 18:00 on the next 2 days after setting bees in the observation hive. We observed randomly selected 10 marked bees in succession. This sequential observation which consisted of 10 individuals was referred to as a Bscan,^ thus one scan needed 1 min (6 s × 10 bees). Each bee was observed for seconds, and its behavior was categorized into four categories: (1) allogrooming (nestmate cleaning), (2) being allogroomed, (3) autogrooming (self-cleaning), and (4) no grooming. We also recorded the body parts being groomed and which legs were used to groom. In this study, we de-fined a grooming to the dorsolateral thorax anteriorly by a middle leg asBthorax-autogrooming.^ We conducted five scans for each colony with more than 3-min inter-vals between scans. These sequential five scans were called aBset.^ A total of seven sets of observations were conducted in each colony for 2 days with more than 1.5-h intervals for each individual set. We conducted 35 and 105 scans of behavior observation in A. cerana and A. mellifera in total, respectively.
2.7. Statistical analyses
All analyses were conducted using the statistical software R 3.1.1 (R Development Core Team2014).
3. RESULTS
3.1. Experiment 1
—susceptibility of bees
less than four days old
The total number of live target bees of
A. cerana and A. mellifera were 53 (survival rate
35 %) and 135 (90 %), respectively. Figure
2
clearly showed that both number of entering adult
mites and infestation rate of trachea were higher in
younger emerged bees irrespective of bee species.
The declines in mite infestation rates with
increas-ing bee age between the two species were similar.
3.2. Experiment 2
—interspecies comparison
of susceptibility
The results are shown in Table
I
. The rate of
infested bees in A. cerana was significantly
higher than that in A. mellifera in cage I and II
assays but not in the case of the hive assay
(Table
I
). The number of mites per trachea in
A. cerana is greater than that in A. mellifera in
all inoculation assays (Figure
3
). There was no
significant intraspecific difference of the number
of mites per trachea in intraspecies among
colo-nies in each assay (p > 0.05, Mann-Whitney or
Steel-Dwass test) except for A. mellifera in the
hive assay (p < 0.05, Steel-Dwass test). Overall,
the ratio of larvae to eggs in A. cerana was
significantly greater than that of A. mellifera in
sugar water in a petri dish
comb with a stand
a cage
mesh cloth
Host bees
Target bees
Figure 1. The experimental layout with a cage. Host bees, target bees, comb on the stand, and sugar water in a plastic petri dish within a cage of mesh cloth were maintained under dark conditions at 32 °C and 55 % RH for 7 days. The petri dish lid has three holes (8 mm diameter) attached to a vinyl chloride tube (15 mm length) to prevent bees from falling into the sugar water.
0% 20% 40% 60% 80% 100% 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 0% 20% 40% 60% 80% 100% 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3
Age (days) of target bees N=38 22 30 10
N=73 78 60 56
Mean female mites per trachea
Infestation rate of trachea
A. cerana A. mellifera a ab b b a ab b b Infestation rate Mean female Infestation rate Mean female
Figure 2. Mean adult tracheal mitesAcarapis woodi found in the large thoracic tracheae of two Apis species, A. cerana (upper ) and A. mellifera (lower ), aged 0–3 days in inoculation assay after 7 days. Columns with the same letter in each honey bee species are not significantly different at 5 % levels by Steel-Dwass test. The data are expressed in means ± SE to show the decline clearly although not parametric. Secondary axis shows the infestation rate of trachea (line chart ).
0 1 2
A. cerana A. mellifera A. cerana A. mellifera
Cage I
Number of mites per trachea
p<0.001 p<0.001 A. cerana A. mellifera Hive Cage II p<0.001 0 2 4 6 8 10 12
a
b
Figure 3. Number of adult mitesAcarapis woodi per the large thoracic trachea in Apis cerana and Apis mellifera in inoculation experiments of cages (a ) and a hive (b ). Boxplots demonstrate the lower quartile, median, and upper quartile, and whiskers represent 1.5 times the interquartile range. The statistical comparisons were performed using a Mann-Whitney U test.
cage II and less than that of A. mellifera in hive
inoculation assays (p < 0.05, chi-square test)
(Table
I
). The mean fecundities of the cage
exper-iments were not different between bee species
(p > 0.05, t test), while in the hive experiment,
the mean fecundity of 3.48 in A. mellifera was
significantly higher than that of 2.20 in A. cerana
(p < 0.001, t test) (Table
I
). There was no
signif-icant intraspecific difference of fecundities among
origins of target bees in each assay (p > 0.05,
ANOVA) except for A. cerana in the cage II assay
(p < 0.05, t test).
3.3. Experiment 3
—interspecies comparison
in frequency of grooming behaviors
There were few records of
Ballogrooming^ and
Bbeing allogroomed^ in our tests. A. mellifera did
more thorax-autogrooming per scan than did
A. cerana , although the frequency of all
autogrooming was higher in A. cerana than in
A. mellifera (Figure
4
).
4. DISCUSSION
Migrating female tracheal mites entered the
tra-cheae of newly emerged bees in A. cerana twice
more frequently than those in A. mellifera .
Considering that the mites infest younger bees more
frequently in both honey bee species and that the
age-related declines of infestation rate of both
spe-cies were similar (experiment 1), A. cerana is more
susceptible to infesting foundress mites than is
A. mellifera . However, fecundities and ratios of
larvae to eggs showed varying values among
experiments and between species. These different
values among experiments may be mainly attributed
to different longevities of host bees. The differences
observed between A. cerana and A. mellifera may
indicate some different symptom for mite infestation
between species, even though showing opposite
results between the cage and hive experiments. For
instance, mite fecundity in A. mellifera was 1.6
times higher than in A. cerana only in the hive
inoculation assay. It is considered that the mite laid
more offspring in the tracheae of A. mellifera or that
the foundress mite in A. mellifera had migrated
further into or out of the trachea as in the cases of
Gary et al. (
1989
) and Mcmullan and Brown (
2005
).
The ratio of larvae to eggs is an indicator of
devel-opmental rate because the time of entering the
tra-cheae is similar between species as seen in
experi-ment 1 and the distribution of migrating female
mites was also not affected by whether tracheae
contained mites or not (Gary and Page
1987
).
There-fore, we expect that the higher fecundity in
A. mellifera and the variation of ratio larvae to eggs
are possibly caused by different broodnest
tempera-ture (Tan et al.
2012
) or some nutritional factor, and
possible explanations warrant further study. In
sum-mary, we propose that more migrating mites enter
the tracheae of A. cerana , and this is a primary
factor causing the explosive epidemic of tracheal
mites in A. cerana , while few mites are observed
in A. mellifera in Japan, even though the use of
Varroa treatments in A. mellifera colonies may be
having some effect in tracheal-mite suppression.
The higher susceptibility to migrating mites
entering into the trachea in A. cerana could help
to explain the imbalance in the host-parasite
rela-tionship. One factor is surely the parasite
prefer-ence to its host. When female mites migrate to
new hosts, they quest and seek out the hosts using
cues of specific hydrocarbons of bee
’s cuticle
(Phelan et al.
1991
). The difference in proportions
of the hydrocarbons between A. cerana and
A. mellifera (Lee et al.
2003
) might influence
0 1 2 3 4 5 6 p<0.05 p<0.01 A. cerana A. mellifera All autogrooming A. cerana A. mellifera Thorax-autogrooming
Number of grooming bees in a scan
the mite preference. Another factor is concerned
with the host resistance to parasitic infestation.
Previous studies in A. mellifera have shown that
resistance to tracheal mites is in part due to
allogrooming and autogrooming to remove the
migrating mites (Danka and Villa
1998
,
2003
;
Pettis and Pankiw
1998
). Our results show that
A. mellifera autogroom their thorax by using their
middle legs twice as frequently as A. cerana ,
indicating that A. mellifera can more efficiently
remove the migrating mites. However, we need to
compare the bee
’s responses to the mite directly as
was done by Pettis and Pankiw (
1998
) and Danka
and Villa (
2003
). The lower frequency of
grooming behavior is likely one cause of the
higher susceptibility to the mite in A. cerana .
Such a lack of a balance in the host-parasite
rela-tionship is seen in A. mellifera and Varroa mite.
A. mellifera shows a higher susceptibility to the
new parasite Varroa mite since A. mellifera has
no effective defense to Varroa mite, while
A. cerana shows a lower susceptibility since
A. cerana has established effective behaviors of
grooming and biting to remove Varroa mite after
a long history of coevolution (Peng et al.
1987
;
Büchler et al.
1992
). Further work is required to
examine in more detail the factors affecting
dif-ferential susceptibility to the tracheal mite in two
honey bee species.
ACKNOWLEDGMENTS
We are grateful to Fumi Konno and Mio Nishiyama of NIES for their assistance in preparing experiments and dissecting bee samples, and to Ayumi Nakamura of NILGS and Akira Suwa and Kunihiko Numajiri in Tsukuba City for providing bee samples. We also thank Koichi Goka, Shigeki Kishi, Toshio Aoki, and our colleagues of NIES and Yoshio Suzuki, Chizuko Yoshida, Akira Kawada, and Jun Arai of the Kawakami F.C. for their kind advice and help. This study was supported by JSPS KAKENHI Grant No. 26290074, the Environment Research and Technology Develop-ment Fund (No. 5-1407) of the Ministry of the Envi-ronment, Japan, and the Sumitomo Foundation.
Différence de sensibilité à l’acarien des trachées Acarapis woodi , entre Apis cerana et Apis mellifera
abeille asiatique / abeille européenne / Acari / c o m p o r t e m e n t d e t o i l e t t a g e / d i f f é r e n c e comportementale
Unterschiedliche Anfälligkeiten bei Apis cerana und Apis mellifera gegenüber der Tracheenmilbe Acarapis woodi
Asiatische Honigbienen / Europäische Honigbienen / Acarapis woodi / Anfälligkeit / Putzverhalten
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